A modified vaccinia Ankara vaccine expressing spike and nucleocapsid protects rhesus macaques against SARS-CoV-2 delta infection

SARS-CoV-2 vaccines should induce broadly cross-reactive humoral and T cell responses to protect against emerging variants of concern (VOCs). Here, we inactivated the furin-cleavage site (FCS) of spike expressed by a modified vaccinia Ankara (MVA) virus vaccine (MVA/SdFCS) and found that FCS inactivation markedly increased spike binding to human ACE2. Following vaccination of mice, the MVA/SdFCS vaccine induced 8-fold higher neutralizing antibodies compared to MVA/S, which expressed spike without FCS inactivation, and protected against the beta variant. We next added nucleocapsid to the MVA/SdFCS vaccine (MVA/SdFCS-N) and tested its immunogenicity and efficacy via intramuscular (IM), buccal (BU) or sublingual (SL) routes in rhesus macaques. IM vaccination induced spike-specific IgG in serum and mucosae (nose, throat, lung, rectum) which neutralized the homologous (WA-1/2020) and heterologous VOCs, including delta, with minimal loss (<2-fold) of activity. IM vaccination also induced both S and N specific CD4 and CD8 T cell responses in the blood. In contrast, the SL and BU vaccinations induced less spike-specific IgG in secretions and lower levels of polyfunctional IgG in serum compared to IM vaccination. Following challenge with SARS-CoV-2 delta variant, the IM route induced robust protection, BU moderate protection and the SL no protection. Vaccine-induced neutralizing and non-neutralizing antibody effector functions positively correlated with protection, but only the effector functions correlated with early protection. Thus, IM vaccination with MVA/SdFCS-N vaccine elicited cross-reactive antibody and T cell responses, protecting against heterologous SARS-CoV-2 VOC more effectively than other routes of vaccination.


Supplementary materials
Detailed materials and methods          Table S1. Lung pathology scores of individual animals at necropsy (day 10 post infection).
Data file S1: Raw Data file ADCP, ADNP and ADCD Assays for monkey sera Antibody-dependent cellular phagocytosis (ADCP), antibody-dependent neutrophil phagocytosis (ADNP) and antibody-dependent complement deposition (ADCD) were measured as previously described (8)(9)(10). Antigen-coupled beads were incubated with appropriately diluted plasma (ADCP 1:200, ADNP 1:50, ADCD 1:10) for 2 hours at 37˚C to form immune complexes. For ADCP, 2.5x10 4 THP-1s cells were added and incubated for 16 hours at 37˚C. For ADNP, leukocytes were isolated from fresh peripheral whole blood and added to immune complexes at 5x10 4 cells/well and incubated for 1 hour at 37˚C. Neutrophils were detected using anti-human CD66b Pacific-Blue. For ADCD, lyophilized guinea pig complement was resuspended, diluted in gelatin veronal buffer with calcium and magnesium (GVB++, Boston BioProducts) and added to immune complexes. The deposition of C3 was detected using an anti-C3 FITC antibody. All functional assays were acquired with an iQue (Intellicyt) and analyzed using Forecyt software. For ADCP, events were gated on singlets and fluorescent cells. For ADNP, bead-positive neutrophils were defined as CD66b positive, fluorescent cells. For both ADCP and ADNP, a phagocytic score was defined as (percentage of bead-positive cells) x (MFI of bead-positive cells) divided by 10000. For ADCD, data was reported as median fluorescence of C3 deposition (MFI).

Antibody-dependent natural killer cell activation (ADNKA)
A modified version of a previously described plate bound antibody-dependent natural killer cell activation (ADNKA) assay was used (11). Wells of a 96 well ELISA plate were coated with 6 μg/ml SARS-CoV-2 spike antigen (2019-nCoV, Sino Biological) in PBS overnight at 4˚C. Nonspecific binding sites were blocked with 5% milk and 4% whey in blotting-grade blocker (BioRad) for 1 hour at RT and 1:100 diluted sera samples added afterwards. PBMCs were isolated from naïve macaques using sodium heparin CPT tubes. Isolated PBMCs were mixed with anti-human CD107a APC-H7 (clone H4A3, BD) in the presence of Brefeldin A and Monensin, and immediately added to the prepared ELISA plate at 5x10^5 cells/well. After 5 hours incubation at 37˚C, cells were stained with anti-CD14 BV605 (clone M5E2), anti-CD8 PerCP-Cy5.5 (clone SK1), anti-NKG2a PE (clone Z199), anti-CD3 BUV395 (clone SP34-2) and Live/Dead Aqua Viability Dye (Life Technologies) in PBS containing 1 mM EDTA for 30 min at RT in the dark. Cells were fixed and acquired. The relative expression of CD107a on LiveCD3-CD14-CD8+NKG2a+ single NK cells was reported.

Cell processing
Post SARS-CoV-2 challenge, samples were processed and stained in BSL-3 facility. For macaques, PBMC from blood collected in sodium citrate CPT tubes were isolated using standard procedures. For processing lymph-node, lymph-node biopsies were dissociated using 70 μm cell strainer. The cell suspension was washed twice with R-10 media. Pelleted cells were suspended in 1ml R10 medium (RPMI(1X), 10% FBS) and stained as described in sections below.

Intracellular Cytokine Staining (ICS) assay
Functional responses of SARS-CoV-2 S1, S2, RBD, and N-specific CD8 + and CD4 + T cells in vaccinated animals were measured using peptide pools and an intracellular cytokine staining (ICS) assay as described previously (1, 2, 12). Overlapping peptides (13 or 17-mers overlapping by 10 amino acids) were obtained from BEI resources (NR-52402 for spike and NR-52419 for nucleocapsid) and different pools (S1, S2, RBD and N) were made. The S1 pool contained peptides mixed from 1-97, the S2 pool contained peptides mixed from 98-181, the RBD pool contained peptides 46-76 and the N pool contained 57 peptides. Each peptide was used at a 1 g/ml concentration in the stimulation reaction. Two million cells suspended in 0.2 ml of RPMI 1640 medium with 10% FBS were stimulated with 1 μg/ml CD28, 1 μg/ml CD49d co-stimulatory antibodies, and different peptide pools. These stimulated cells were incubated at 37°C in a 5% CO2 conditioned incubator. After 2 hrs of incubation, 1 μl Golgi-plug and 1 μl Golgi-stop/ml were added and samples were incubated for 4 more hours. After a total 6 hours of incubation, cells were transferred to 4 o C overnight and stained the next day. Cells were washed once with FACS wash (1X PBS, 2% FBS and 0.05% sodium azide) and surface stained with Live/Dead-APC-Cy7, anti-CD3, anti-CD4 and anti-CD8, each conjugated to a different fluorochrome for 30 minutes at RT. The stained cells were washed once with FACS wash and permeabilized with 0.2 ml of Cytofix/Cytoperm for 30 minutes at 4 o C. Cells were washed once with perm wash and incubated with anti-cytokine (IFNγ, TNFα, IL-2, IL-4, and IL-17) antibodies for 30 minutes at 4 o C. Finally, the samples were washed once with perm wash and once with FACS wash and fixed in 4% paraformaldehyde for 20 minutes before acquisition on a BD LSR Fortessa flow cytometer. Data was analyzed using FlowJo software.

Viral RNA extraction and quantification
SARS-CoV-2 subgenomic RNA was quantified in nasopharyngeal swabs, and broncho-alveolar lavages (BAL) as described previously (1, 2). Quantitative reverse transcription PCR (qRT-PCR) was performed on the subgenomic mRNA transcript of the E gene (13). Swabs were placed in 1ml of Viral Transport Medium (#Cat VR2019-1L, VTM Labscoop). Viral RNA was extracted from NP swabs, and BAL on fresh specimens using the QiaAmp Viral RNA mini kit according to the manufacturer's protocol. Quantitative reverse transcription PCR (qRT-PCR) was performed on the subgenomic mRNA transcript of the E gene (13) using primer and probe sequences for are SGMRNA-E-F: and SGMRNA-E-Pr: 5'-FAM-ACACTAGCCATCCTTACTGCGCTTCG-3'. qPCR reactions were performed in duplicate with the Thermo-Fisher 1-Step Fast virus mastermix using the manufacturer's cycling conditions, 200nM of each primer, and 125nM of the probe. The limit of detection in this assay was about 128 copies per ml of VTM/BAL depending on the volume of extracted RNA available for each assay. To verify sample quality, the CDC RNase P p30 subunit qPCR was modified to account for rhesus macaque specific polymorphisms. The primer and probe sequences are RM-RPP30-F 5'-AGACTTGGACGTGCGAGCG-3', RM-RPP30-R 5'-GAGCCGCTGTCTCCACAAGT-3', and RPP30-Pr 5'-FAM-TTCTGACCTGAAGGCTCTGCGCG-BHQ1-3' (14). A single well from each extraction was run as described above to verify RNA integrity and sample quality via detectable and consistent cycle threshold values (Ct between 25-32).